Dental pain is one of the most prevalent types of orofacial pain but its mechanism is still poorly understood. Understanding cellular and molecular mechanisms of tooth pain is critical for developing novel strategies for better treating dental pain. Nociceptors are divided into largely two populations, peptidergic nociceptors and non-peptidergic nociceptors. In tooth pulp, a large population of afferents is known to be peptidergic. However, there is no convincing evidence of a pulpal projection of IB4-positive non-peptidergic afferents. Our preliminary study using a novel mouse model revealed that tooth pulp is densely innervated by a subpopulation of nociceptive primary afferents selectively expressing Mrgprd, Mas-related G protein-coupled receptor subtype D. Since the Mrgprd-positive afferents represent a majority of non-peptidergic nociceptors in skin, it strongly suggests that tooth pulp has a profuse projection of non-peptidergic nociceptors. In this application, we will define the neurochemical properties of Mrgprd-positive dental afferents in retrogradely labeled trigeminal ganglia to provide unambiguous evidence of the pulpal projection of IB4-positive non-peptidergic afferents (Aim I). In order to demonstrate that the afferents are involved in neural responses and potentially pain upon tooth injury or inflammation, we will demonstrate the involvement of Mrgprd-positive afferents in the activation of trigeminal nociceptive circuitry upon tooth injury (Aim II) and investigate whether tooth inflammation enhances ATP-evoked activation of Mrgprd-positive pulpal nociceptors (Aim III). These studies will provide strong anatomical and functional evidence of a novel subset of dental afferents and provide the scientific rationale for developing potential analgesic agents targeting molecules enriched in this subpopulation of neurons. PUBLIC HEALTH RELEVANCE: Dental pain is a major health problem. This study aims to elucidate the role of a novel type of nerve fibers conveying pain signals from tooth. The positive outcome of this study will help to better understand the cellular and molecular mechanisms of dental pain and provide a rationale for developing a strategy for preventing and treating this condition.